Cell death is an essential aspect in the life of a multicellular animal, playing an integral role in cell turnover, embryonic development, and responses to environmental stress. The long term objective of this proposal is to gain an understanding of the molecular basis by which programmed cell death (PCD) and programmed cell removal are regulated in a unique model system, the colonial urochordate Botryllus schlosseri, and how these processes are counterbalanced with stern cell-derived organismal regeneration. In this animal, PCD occurs in cyclical fashion every week, during which all adult individuals in a colony die synchronously by apoptosis within 24 hours. During this period, cell corpses from adult tissues are engulfed by circulating phagocytic cells, and a new asexual generation of adults simultaneously replaces the old one as it is resorbed. The proposed studies will initially seek to identify components of the cell death machinery that are expressed during PCD/removal in B. schlosseri colonies, and how developing buds regulate their activities (aim #1). Caspase-3, a cysteinyl protease, is the principal executioner caspase in mammalian cells. The proposed studies will seek to clone the B. schlosseri homologue of this gene using degenerate polymerase chain reaction technology. Determining the complete nucleotide and amino acid sequence of the caspase-3 gene will pave the way to investigate its expression pattern, substrate specificity and function in B. schlosseri colonies (aim #2). Recent findings from this laboratory have strongly suggested that Botryllus phagocytes may be critical effectors in homeostatic regeneration of tissues and new individuals, by functioning as recycling shuttles during the death phase of a B. schlosseri colony. Aim #3 will seek to investigate phagocytic function in this animal. Specifically, india ink and phosphatidylserine receptor-specific antibodies will be used to track phagocyte homing patterns during and following PCD and removal. Liposome-encapsulated dichloromethylene diphosphonate will also be used to selectively deplete circulating phagocytes. Lastly, DNA and protein-specific fluorescent tracers will be used to selectively label degenerating adult tissues and monitor their translocation and incorporation within developing buds.